Enhancing anti-tumor immune responses using cytokines that have potent and pleiotropic effects has great potential to improve cellular immunotherapy. Unfortunately, as a result of these potent functional properties, systemic delivery of cytokines often has severe side-effects that have greatly limited their use and efficacy. Local expression of cytokines achieved experimentally has shown remarkable effects but it is extremely difficult to treat tumors that have already spread and are not easily accessible. We are developing a novel fusion protein (FP) strategy that combines these approaches whereby we deliver a cytokine systemically so that it might reach disseminated tumors, but have it function preferentially at the tumor thus minimizing unwanted side effects. In this proposal, we are developing a novel approach that employs a FP in which a cytokine is joined to a specific binding moiety (such as a single-chain Variable Fragment (scFv)), separated by a protease site. Before cleavage, the cytokine is largely inactive since it is bound to the inhibitory scFv, but after cleavage by proteases preferentially expressed at the tumor site, the cytokine becomes biologically available to interact with dramatically higher affinity receptors on immune cells. The novel protease activated cytokine approach we are developing is fundamentally different than previous approaches to increase cytokine at tumor sites. Mechanistically, this strategy employs a scFv not to target the cytokine to the tumor but to inhibi cytokine activity until it becomes activated by proteases that are over expressed in the tumor microenvironment. We are characterizing activatable FPs that contain either IL-2, IL-12, or the chemokine CXCL10, that are designed to overcome two important barriers to successful immunotherapy: the immunosuppressive microenvironment of many tumors and the often inefficient homing of T cells to tumor sites. We have incorporated a matrix metalloproteinase (MMP) site in the FPs as proof of concept since MMPs are often functionally over expressed in the tumor microenvironment and intimately involved in tumor progression. This approach is versatile since it can be customized to virtually any protease that is preferentially expressed at tumor sites by substituting different protease sites in the FP. Further, given the immense diversity of scFv that can be isolated using phage display, we hypothesize this approach could be used for essentially any cytokine or chemokine. After validating their activity in vitro, we wil employ an integrated approach to examine how delivery of these fusion proteins affect the tumor microenvironment and the anti-tumor immune response using the B16 melanoma model (which are relatively poorly infiltrated with CD8 cells) and the Colon 38 tumor model (which have numerous CD8 cells but which function suboptimally). By using a combination of flow cytometric analyses, functional analyses, qRT- PCR, as well as whole mount histology and other imaging technologies, we will gain a comprehensive insight of the effects of fusion proteins in vivo and their mechanism of action. If successful, this approach has the potential to create an entirely new class of biologics for tumor therapy.